Diagnosis of alpha 1-antitrypsin deficiency by enzymatic amplification of human genomic DNA and direct sequencing of polymerase chain reaction products.

We have compared sequencing of cloned "polymerase chain reaction" (PCR) products and the direct sequencing of PCR products in the examination of individuals from six families affected with alpha 1-antitrypsin (AAT) deficiency. In families where paternity was in question we confirmed consanguinity by DNA fingerprinting using a panel of locus-specific minisatellite probes. We demonstrate that direct sequencing of PCR amplification products is the method of choice for the absolutely specific diagnosis of AAT deficiency and can distinguish normals, heterozygotes and homozygotes in a single, rapid and facile assay. Furthermore, we demonstrate the reproducibility of the PCR and a rapid DNA isolation procedure. We have also shown that two loci can be simultaneously amplified and that the PCR product from each locus can be independently examined by direct DNA sequencing.     

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

Summary The prevalence of the 281 (GlyGlu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.     

Recovery of murine lens epithelial cells from single and fractionated doses of X rays and neutrons

Subpopulations of mouse lens epithelial cells, differing in proliferative status, were irradiated with either X rays or fission spectrum neutrons given singly or in four weekly fractions. After various times, epithelia were mitogenically stimulated by wounding and DNA synthesis responses were determined by incorporation of [3H]thymidine. At 1 h following both X and neutron irradiations, significant suppression of the wound response after single doses and a sparing effect of fractionation were evident in both the mitotically quiescent and the slowly proliferating subpopulations. At 1 week following single or fractionated doses of both radiations, recovery was evident in both subpopulations. By 4 weeks, the quiescent subpopulation showed significant recovery after both single and fractionated doses of X rays or neutrons. In contrast, a marked decreased ability to respond after neutron irradiation and, in addition, a significant enhancement effect of neutron fractionation were observed for the slowly proliferating subpopulation. Per gray, neutrons were about 7.5 times more effective than X rays as a single dose and 25 times more effective in four equal fractions. The shift from an initial sparing to a final enhancing effect of neutron fractionation for the slowly proliferating subpopulation has importance for understanding divergent early and late radiation responses following dose fractionation.     

Studies on the host/parasite interface during the development of a pentastomid arthropod parasite in rodent intermediate hosts, with observations on protective surface membranes

The changing structure of the cuticle of the arthropod pentastomid parasite Porocephalus crotali, during growth to the infective stage in mouse and rattlesnake hosts, is described. The outermost cuticulin layer of the cuticle in instars II-VI is elevated to form a dense mat of epicuticular hairs. Since the VI larval cuticle is retained by the infective (VII) nymph as a protective sheath, effectively all stages in mice present a hairy surface to the host and this may constitute a physical barrier to inflammatory cells. The entire surface is overlain by a triple-track 'unit' membrane whose biophysical properties resemble those of a conventional plasma membrane, and there is evidence to suggest that this membrane is susceptible to immune attack. Under natural circumstances, epicuticular hairs entrap secretion, delivered to the cuticle via innumerable minute ducts which communicate with tegumental secretory cells termed subparietal cells (SPC). SPC synthesize lamellate droplets which unfold on the cuticle to constitute a layer of protective polymorphic vesicles. By contrast, infective nymphs in snakes possess a smooth cuticle and SPC membranous secretion is stacked over the entire surface, in sheets up to 20 deep. The function of the lipid and protein components of SPC secretion is discussed.     

Fine structural aspects of secretory processes in a pentastomid arthropod parasite in its mouse and rattlesnake hosts

The histology and development of three extensive glands in the porocephalid pentastomid Porocephalus crotali is described by light and electron microscopy, during growth of the parasite to an infective stage in the tissues of mouse; the infective stage in rattlesnake definitive hosts is also included. These glands elaborate excretory/secretory components which are channelled, via chitin-lined efferent ductules, on to the parasite cuticle. Hook and frontal glands are relatively compact, and within each gland ductules serving individual secretory lobules collect into common ducts which discharge over each of the four hooks, or at the anterior margin of the cephalothorax respectively. Subparietal gland cell lobules, composed of two large and two small secretory cells, are distributed under the cuticle and each is served by a single efferent ductule; these erupt over the entire cuticle. The large cells in subparietal glands secrete lamellate droplets which coat the cuticle with thin layers. Identical cells are found in hook and frontal glands, in addition to to three morphologically distinct types of protein secretory cell. Preliminary data on the composition and immunological properties of the various secretory products are presented.     

Contribution of midbody channels to embryogenesis in the mouse

Summary We have examined the persistence of midbody channels during the second, third, and fourth cleavage cycles of the mouse using immunofluorescence to map the distribution of midbody microtubule bundles in intact embryos. Electron microscopy showed these bundles to be a characteristic feature of midbodies throughout the interphase period. In recently-divided embryos at each cleavage stage the number of midbodies was half the number of blastomeres, and declined towards zero as the next cleavage approached. This indicated to us that the only midbodies present in each stage were those which had arisen in the immediately-preceding division. Of those blastomeres which were in mitosis at the time of fixation, less than 4% were connected via a midbody to another blastomere, demonstrating that persistence of midbodies beyond a single cleavage cycle is a rare event. We conclude that midbody channels in our embryos are likely to connect only pairs of sister blastomeres because midbodies do not persist through multiple cleavage cycles. Midbody channels cannot, therefore, be regarded as providing extensive cell coupling in advance of the onset of gap junctional communication.     

In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

Recruiting physicians to rural practice. Suggestions for success.

Medical school graduates from 1986 to 1988 and current residents in 12 family practice residency programs in the Northwest (N = 302) were surveyed to identify important factors in the recruitment process for their first postresidency placement. The study sought to compare the recruitment practices of rural communities and urban sites. Specific questions addressed in the study concerned sources of information about practice opportunities, stage in training when job search was initiated, factors related to unsuccessful site visits, and activities scheduled in the visit. Results indicated that referrals from faculty were the most valued source of information. Most job searches were initiated in the first 6 months of the third year in training. An unreceptive physician community and a reluctant spouse or partner were substantial problems for residents making site visits to rural communities. Rural sites tended to provide a broader mix of professional and personal activities during the visit.     

The immune recognition of malaria antigens

Owing to the demonstration that the immune response of inbred mice to some defined malaria antigens is influenced by the major histocompatibility complex (MHC), and the finding that only a minority of individuals living in malaria-endemic areas appear to recognize such antigens, there are fears that synthetic subunit malaria vaccines will be poorly immunogenic in a substantial proportion of the target population. Such fears have been reinforced by the results of the first two human malaria vaccine trials. In this review Eleanor Riley, Olle Olerup and Marita Troye-Blomberg summarize the experimental evidence for MHC-related genetic restriction of malaria immunity and discuss some alternative explanations for nonresponsiveness in populations living in malaria-endemic areas.